While individual genes, regulatory factors, and events in the mammalian nucleus have been intensely studied and are beginning to be understood, the mechanisms by which the nucleus is organized and nuclear processes are coordinated remain largely unknown. Recently, the study of nuclear structure and function has taken on increased medical relevance with the discovery that A-type nuclear lamins, core constituents of the nuclear substructure, are targets for mutation in a wide variety of human diseases including dystrophic and progeroid syndromes (collectively termed laminopathies). An important question in understanding the molecular basis of the laminopathies is how mutations throughout the LMNA coding region could be associated with tissue-specific disorders. We have established a functional association between A-type lamins and the retinoblastoma protein (pRB), a known differentiation factor, regulator of cell proliferation and tumor suppressor. Lamin A/C protects pRB from proteosome-mediated degradation, pRB is required for normal differentiation of muscle and fat tissue, two of the tissues frequently affected in laminopathies. Moreover, pRB has been implicated in cellular senescence, a tissue culture model system for aging. A major focus of this proposal is to characterize the interactions between A-type lamins and pRB at a molecular and cellular level, and to determine whether lamin-dependent regulation of pRB is functionally significant in cell proliferation, stable tissue differentiation and the pathogenesis of laminopathies. We view the functional interaction between lamins and pRB as a prototype for how lamins coordinate nuclear regulatory proteins. Both directed and unbiased approaches will be initiated to identify novel, functionally significant lamin A/C interacting proteins. In vitro cell differentiation assays have served as important tools to identify key regulatory molecules that mediate cell specification. Given that A-type lamin expression is generally restricted to differentiating tissues and LMNA mutations cause dystrophic syndromes primarily affecting muscle and fat tissue, as a final Aim, a project has been initiated to examine the role of A-type lamins for myocyte and adipocyte differentiation. Myoblast cell lines, generated from Lmna-/- mice, exhibit differentiation defects. These defects will be studied at the molecular level to better understand how aberrant A-type lamin function leads to tissue degeneration.